The antibody response to HIV-1 that is mediated by B-1 B cells is a largely unexplored area in HIV vaccine development. However, recent progress has raised important questions about whether the conventional B-2 B cell-oriented immunization strategies are solely responsible for induction of HIV-1 broadly neutralizing antibodies (bnAbs), [including those that are highly specific for carbohydrate epitopes and those that are characteristically polyreactive and autoreactive, as these antibodies are often seen in the B-1 repertoire.] Importantly, such antibodies represent dominant classes of HIV-1 bnAbs identified from a portion of HIV-infected subjects using the advanced technologies of single-cell cloning and deep sequencing. Our team has been studying carbohydrate antigens and anti-carbohydrate B cell responses for years. Recently, we obtained evidence that oligomannose-based HIV antigens are immunogenic in vivo under certain conditions. In essence, we observed that autoantibodies targeting Man9 clusters were significantly increased in the blood circulation of men with aggressive prostate cancer, in the cerebrospinal fluid of patients with multiple sclerosis, and in the serum of mice with experimental autoimmune encephalomyelitis (EAE). Interestingly, co- immunization of SJL/J mice with a Man9-KLH conjugate at the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. These anti-sera are strongly cross-reactive with HIV-1 gp120 glycoproteins (see Preliminary Results). Moreover, we discovered that immunization of non-human primates (NHP) using human RV144 HIV vaccines elicited significant levels of antibody responses to broadly HIV- neutralizing glyco-epitopes, including oligomannoses and other N-glycan cryptic antigens (Preliminary Results). Most importantly, we recently uncovered a significant population of oligomannose (Man9) cluster-binding B cells (BMan9+) in the naive B cell repertoire that are readily reactive with oligomannose clusters. These B cells represent approximately 12% of CD19+CD45+ B cells in the blood circulation of healthy human subjects. Interestingly, the BMan9+ B cell population is also present in mice, constituting approximately 10% of B-1a (CD19+CD5+) and 2% of B-1b (CD19+CD5-) B cells in the peritoneal cavity. Given that B-1 and B-2 B cells differ significantly in responding to an antigenic stimulation and that most HIV-1 vaccine strategies were developed based on B-2 responses without consideration of the B-1 potential, we propose to conduct a focused investigation of the B-1 B cell-mediated antibody responses to HIV-1. [In essence, we will apply a key HIV vaccine, RV144, to probe the B-1/ BMan9+ B cell responses in multiple species, including human, NHP, IgH-allotype chimeric mice, and humanized mice. Specifically, we will examine whether human RV144 vaccine elicited the characteristic profile of anti-carbohydrate antibody responses as observed in NHP models (Aim 1); examine contributions of murine B-1 and B-2 B cells to production of anti-HIV-1 antibodies in responding to RV144 vaccines (Aim 2); determine whether the human BMan9+ B cells in humanized mice contribute to anti-oligomannose responses and other anti-HIV antibody responses (Aim 3); and explore potential molecular signatures of B-1/BMan9+ B cell-mediated anti-carbohydrate antibody responses by a comparative IgV region sequence analysis (Aim 4).] [In summary, this revised R01 application focuses on identification of molecular and cellular signatures of the B-1/BMan9+ B cell-mediated immune responses to HIV-1 and exploration of the potential of this newly recognized B cell subset in human HIV vaccine development. If successful, this project could lead to the strategic development of novel preventive and/or therapeutic vaccine strategies against HIV infection and AIDS.]